A wide variety of biologically active proteins and polypeptides can now be produced in sufficiently large quantities for use as drugs. For example, the development of the hybridoma method and recombinant DNA techniques have made it possible to produce antibodies on large scale. This has allowed wide use of pharmaceutical compositions containing proteins, such as antibodies, for treating a variety of diseases. Such treatments normally require administering to a patient the proteins at high concentrations.
However, a protein that has desired therapeutic properties may not have sufficiently high solubility. Even for those proteins that have high solubility, high concentration liquid formulations tend to have short shelf lives and may lose biological activity as a result of chemical and physical instabilities during the storage. Additionally, proteins are generally more viscous at high concentrations, which can complicate packaging, storage and delivery of the protein therapeutic. Furthermore, chemical instability may be caused by deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange, and physical instability may be caused by protein denaturation, aggregation, precipitation or adsorption. Among those, aggregation, deamidation and oxidation are known to be the most common causes of antibody degradation (Wang et al., 1988, J. of Parenteral Science & Technology 42 (Suppl.):S4-S26; Cleland et al., 1993, Critical Reviews in Therapeutic Drug Carrier Systems 10(4):307-377; Manning et al., 1989, Pharm. Res. 6:903-918). Aggregate formation during storage of a liquid protein composition can adversely affect the biological activity of the protein, resulting in loss of therapeutic efficacy and/or an increase in immunogenicity in humans. Aggregate formation may also cause other problems such as blockage of tubing, membranes, or pumps when the protein composition is administered using an infusion system.
Due to the instability of proteins in liquid pharmaceutical formulations, protein therapeutics are often packaged in the lyophilized form along with a suitable liquid medium for reconstitution. Although lyophilization improves storage stability of the composition, many proteins exhibit decreased activity, either due to storage in the dried state (Pikal, 1990, Biopharm. 27:26-30) or as a result of aggregate formation or loss of catalytic activity upon reconstitution as a liquid formulation (see, for example, Carpenter et al., 1991, Develop. Biol. Standard 74:225-239; Broadhead et al., 1992, Drug Devel. Ind. Pharm. 18:1169-1206; Mumenthaler et al., 1994, Pharm. Res. 11:12-20; Carpenter et al., 1988, Cryobiology 25:459-470; and Roser, 1991, Biopharm. 4:47-53). Lyophilized formulations of antibodies also require a prolonged process for lyophilization and high cost for manufacturing. A lyophilized formulation has to be reconstituted aseptically and accurately by healthcare practitioners prior to administering to patients. The reconstitution procedure is cumbersome and the time limitation after the reconstitution can cause great inconveniences in administering the formulation to patients, leading to significant waste, if not reconstituted properly or if the reconstituted dose is not used within six (6) hours and must be discarded. Reconstitution may also increase the possibility of incorrect dosing. Thus, a lyophilized formulation which is more stable and is readily reconstituted with little loss in potency is desirable.
A desirable alternative to lyophilized formulations is liquid formulations of protein therapeutics having concentrations comparable to or higher than the reconstituted lyophilized formulations. Such liquid formulations of protein therapeutics can be administered to a subject without the need of reconstitution, thereby allowing healthcare practitioners much quicker and easier administration of protein therapeutics to a patient. In addition, the manufacturing process of the liquid formulation protein therapeutics is simpler and more efficient than the manufacturing process for the lyophilized version because all stages of the manufacturing of the liquid formulations are carried out in an aqueous solution, involving no drying process, such as lyophilization. Accordingly, it is also more cost effective. The development of high concentration, ready-to-use, liquid formulations of protein therapeutics has thus attracted great attention in the biopharmaceutical industry.
Stability of proteins and polypeptides in pharmaceutical formulations, both liquid and lyophilized, can be affected, for example, by factors such as pH, ionic strength, temperature, repeated cycles of freeze-thaw, and exposure to mechanical shear forces such as occur during processing. Various highly stable, high concentration liquid formulations have been successfully developed. For example, liquid formulations of antibodies that are stable for more than 5 years when stored at 4° C. have been reported. U.S. Pat. No. 6,525,102 discloses a stabilized liquid polypeptide-containing pharmaceutical composition. The composition comprises an amino acid base, which serves as the primary stabilizing agent of the polypeptide, and an acid and/or its salt form to buffer the solution within an acceptable pH range for stability of the polypeptide. The compositions are near isotonic. The '102 patent also discloses methods for increasing stability of a polypeptide in a liquid pharmaceutical composition and for increasing storage stability of such a pharmaceutical composition. Lyophilized formulations are common and their stability and reconstitution characteristics may be modified by the addition of stabilizers and/or excipients. However, the development of such liquid and lyophilized formulations depends on the particular protein therapeutics, and often requires significant optimization efforts. Thus, improving the stability of pharmaceutical compositions containing protein therapeutics of a pharmaceutically effective concentration remains a challenge. An additional challenge is providing for formulations which have low enough viscosity to be readily manufactured and/or administered at high concentrations.
In a traditional approach to therapeutic protein (e.g., antibody) development, the protein that has a desired activity and/or property (e.g., binding affinity) is first generated. The protein is then submitted for formulation development to determine the optimal formulation and storage conditions. Traditional screening and optimization processes require lengthy stability studies which are both time consuming and can only examine a limited number of potential formulations. If the protein should fail to meet the formulation requirements, the whole drug development process essentially fails. Thus, there is a need for methods that allow incorporating formulation considerations into early stages of the drug development process. In addition, since the desired shelf life can be as long as one year or longer, methods relying monitoring the formulations in real time are inefficient. Attempts have been made to develop methods for rapidly screening formulations. For example, U.S. Pat. No. 6,232,085 discloses a multi-variable method for optimizing the shelf life of a protein which is capable of denaturing due to a thermal change. The method comprises contacting the target molecule with one or more of a multiplicity of different molecules or different biochemical conditions in each of a multiplicity of containers, simultaneously heating the multiplicity of containers, measuring in each of the containers a physical change associated with the thermal denaturation of the target molecule resulting from heating, generating a thermal denaturation curve for the target molecule as a function of temperature for each of the containers, comparing each of the denaturation curves to (i) each of the other thermal denaturation curves and to (ii) the thermal denaturation curve obtained for the target under a reference set of biochemical conditions, and ranking the efficacies of multiplicity of different molecules or the different biochemical conditions according to the change in each of the thermal denaturation curves. However, such methods are cumbersome and do not address the address the intrinsic properties or characteristics, such as, pI and Tm, of the protein to be formulated.
The intrinsic properties of proteins not only affect their formulation characteristics but may have serious implications for their therapeutic use as well. For example, studies have shown that recombinant toxins made up of a cell-targeting Fv portion of an antibody fused to a toxin have non-specific dose limiting toxicities (e.g., non-specific liver toxicities) which are attributable to the high isoelectric point (pI) of the Fv portion of the molecule. Lowering the pI of the Fv portion of these recombinant toxins by site directed mutagenesis reduced their non-specific toxicity in animal models without altering reducing their antitumor activity (Onda et al., 1999, J. Immunol., 163: 6072-77, Onda et al., 2001, Cancer Res., 61: 5070-77). Likewise, lowering the pI of a radiolabelled anti-tumor dsFv (disulfide stabilized Fv) by chemical modification increased renal clearance thereby decreasing the buildup of radioactivity in the kidney (Kim et al., 2002, Nucl. Med. Biol., 29: 795-801).
In other studies the transendothelial migration and endocytosis of antibodies was enhanced by cationization to increase the pI. The cationized antibodies retained their binding affinity and were rapidly internalized into cells with minimal non-specific toxicity or immunogenicity (Pardrige et al., 1998, J. Pharmaol. Exp. Therap., 286: 548-54). Cationization of antibodies has also been shown to enhance the delivery of antibodies across the blood brain barrier (Triguero et al., 1989, PNAS, 86: 4761-4765) These data indicate that there may be an optimal pI for certain therapeutic proteins, such as antibodies or chimeric proteins made up of antibody domains. Particularly those proteins which carry a toxin, are required in large doses for optimal therapeutic response or those which are required intracellularly or in extravascular compartments.
Thus, there is a need for more efficient methods that allow quick indication of the shelf life and/or clinical properties of protein formulations based on the intrinsic properties of the protein. Additionally, because certain intrinsic properties, such as pI and Tm, are generally not selection criterion when therapeutic proteins (e.g., antibodies) are developed, proteins which are therapeutically active may have suboptimal formulation or clinical properties. Methods to engineer certain intrinsic properties, such as pI and Tm, downstream of, or concurrently with development would allow the rapid production of proteins with preferred formulation and therapeutic characteristics.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.